1. Field of the Invention
This invention relates to a method for detection of anencephaly using a nucleic acid coding for a protein kinase C substrate. In particular, the protein kinase C substrate is a member of the MARCKS family of protein kinases.
2. Description of the Related Art
When macrophages encounter Gram negative bacteria a number of protein kinase C (PKC)-dependent signal-transduction pathways are activated. These lead to the cytoskeletal rearrangements necessary for phagocytosis and migration, the release of inflammatory mediators such as prostaglandins and leukotrienes, and the secretion of hydrolytic enzymes and reactive oxygen intermediates. Bacterial lipopolysaccharide (LPS) or endotoxin, the major surface component of gram-negative bacteria, has a profound modulatory effect on PKC-dependent responses. LPS alone does not activate PKC, but it primes macrophages for vastly potentiated responses when the cells subsequently encounter PKC-activating agonists. Concomitant with priming, LPS induces the synthesis of two myristoylated proteins with apparent molecular masses of 68K and 42K, respectively.
LPS or endotoxin, a major surface component of Gram negative bacteria is a potent activator of cellular and humoral immunity. LPS enhances the tumoricidal activity of macrophages and stimulates the release of numerous inflammatory mediators such as tumor necrosis factor, arachidonic acid metabolites, complement components, reactive oxygen intermediates, nitric oxide, and hydrolytic enzymes.
Much has been learned recently about the mechanism by which cells recognize LPS. LPS associates with LPS binding protein, and the resulting complex promotes the binding of LPS to CD14 on the cell surface. CD14 appears to be a subunit of a multicomponent LPS receptor. A 70 kDa membrane protein, the scavenger receptor, and the CD11b/CD18 complex have all been demonstrated to bind LPS, but the significance of this is not known. A recent report has cast doubt on the identity of the 70 kDa protein as an LPS receptor. Binding of LPS to CD14 causes rapid protein tyrosine phosplhorylation of a number of intracellular proteins that include three members or the MAP kinase family (MAPK1, MAPK4 and p38). p38 is homologous to the S. cerevisiae HOG1 gene product, which is involved in osmotic regulation. Data from a number of laboratories indicate that protein kinase C (PKC) induced phosplhorylation is necessary for a full functional response to LPS. LPS primes macrophages for enhanced PKC mediated responses, and promotes the synthesis of two PKC substrates, MARCKS and MacMARCKS. Because LPS treatment also potentiates the phosplhorylation of these proteins, they are ideal candidates as effectors of LPS-induced responses.
LPS induces profound changes in gene expression in macrophages, often by activation of the NF.kappa.B transcription factor. Recent investigations into the promoter region of the i-NOS gene have demonstrated two regions which are required for LPS-dependent transcription, and both of these regions contain NF.kappa.B binding sites. However, the NF.kappa.B binding sites were found by computer analysis of the region, and the participation of NF.kappa.B in transcriptional activation of i-NOS was not confirmed by site directed mutagenesis. Investigation into the 5' upstream sequence of another LPS responsive gene, Rantes, demonstrated the importance of an NF.kappa.B binding site, as well as the 3' half of the proximal AP1 site. It is therefore clear that a great deal of complexity exists in the transcriptional activation of LPS responsive genes.
The 68K protein induced by LPS, myristoylated alanine-rich protein kinase C substrate (MARCKS), is a major cellular substrate of PKC that binds calmodulin and has a role in diverse cellular processes including macrophage and neutrophil activation, mitogenesis, and neurosecretion, and may regulate the reversible attachment of the actin cytoskeleton with the substrate-adhlerent plasma membrane in motile phagocytes. (Li et al (1992) Cell 70:791-801).
PKC phosphorylation produces translocation of MARCKS from membrane to cytoplasm in many cells. The binding of MARCKS to biological membranes appears to require both hydrophobic insertion of its myristoyl chain into the lipid bilayer and electrostatic interaction of its basic domain with acidic lipids (Kim et al (1994) J. Biol. Chem. 269:28214-28219). The amino-terminal glycine of MARCKS is myristoylated, and the protein is rich in acidic residues except for one basic region. The murine protein for example contains a cluster of 13 basic and 0 acidic residues between amino acids 145 and 169. This basic region is highly conserved, contains the only serines phosphorylated by PKC, binds calmodulin (CaM) in a calcium-dependent manner, and binds actin filaments (Kim et al, 1994).
In addition to hydrophobic and electrostatic interactions with phospholipid bilayers, there is good experimental evidence that the binding of MARCKS to biological membranes also involves specific protein-protein interactions. The punctate distribution of MARCKS in macrophage membranes suggests the protein also interacts with cytoskeletal elements. These protein-protein interactions need not be strong because once MARCKS is bound to the bilayer component of the membrane via its myristoyl chain and basic domain, even a weak protein-protein interaction would suffice to create a punctate distribution. Weak protein-protein interactions may also target MARCKS to specific membranes (e.g., the plasma rather than Golgi membranes) (Kim et al, 1994).
The cDNA encoding MARCKS has been cloned and sequenced from a number of species. The actual molecular mass of MARCKS, calculated from its primary structure, ranges from 28 kd to 31 kd, while its apparent molecular mass determined by SDS-PAGE varies from 67K to 87K. This anomalous migration on SDS gels can be ascribed to the high axial ratio and rod-shaped dimensions of MARCKS. Comparison of the sequences reveals that MARCKS contains two highly conserved domains: an N-terminal domain that contains the myristoylation site and which appears to function in membrane binding, and an effector domain, located in the middle of the rod-shaped protein, that contains all the serine residues known to be phosphorylated, as well as the calmodulin and actin binding sites. The proximity of the phosphorylation sites to the actin and calmodulin binding sites explains the reciprocal regulation of phosphorylation and the binding of these two proteins (Li et al, 1992).
The 42K myristoylated protein induced during LPS priming is a PKC substrate that shares the effector domain of MARCKS, but has a distinct N-terminal membrane-binding domain. Like MARCKS, this 42K protein is an alanine-rich protein that binds calmodulin in a manner regulated by PKC. Since the 42K protein is structurally and functionally related to MARCKS and since it is predominantly expressed in LPS-stimulated macrophages, it has been named MacMARCKS.
MacMARCKS, like MARCKS, is heat stable. MacMARCKS has been cloned from an LPS-induced rabbit alveolar macrophage cDNA library. The transfected rabbit cDNA encodes a myristoylated protein that migrates on 2D IEF-SDS-PAGE with an apparent molecular mass of 42K and a pI of 4.2, as does the in vitro translated protein (Li et al, 1992).
The rabbit MacMARCKS protein sequence shares a 92% identity with the murine protein sequence. Moreover, a comparison of rabbit and murine MacMARCKS with human, bovine, murine, rat and chicken MARCKS reveals a similar domain structure. Both have myristoylated N-termini that differ in amino acid sequence but contain similar charge distributions: two positive charges followed by two negative charges. The myristoylation domain is followed by two regions of major homology (MH1 and MH2). However, there is an important difference between MARCKS and MacMARCKS: the sequence FKKS (SEQ ID NO:1) that comprises the second of 2 phosphopeptides of MARCKS is FKKP (SEQ ID NO:2) of MacMARCKS, accounting for the absence of a phosphopeptide 2 in phosphorylated MacMARCKS.
Several lines of evidence suggest that MARCKS and MacMARCKS are members of a protein family. Both are acidic, myristoylated PKC substrates with similar and unusual amino acid compositions: alanine, glycine, proline and glutamic acid comprise approximately 60% of the total amino acids of both proteins. Both proteins have a similar domain structure: an N-terminal myristoylated domain, a highly conserved MH2 domain, and a basic effector domain that contains the phosphorylation sites and the calmodulin binding site.
Both MacMARCKS and MARCKS contain the myristic acid moiety in an amide linkage to an N-terminal glycine residue. Myristoylation is absolutely required for membrane binding of a number of important signal-transducing molecules including MARCKS, certain a subunits of the heterotrimeric G-proteins, and the src family of tyrosine kinases. Evidence suggests that myristoylated proteins do not associate with membranes by the mere insertion of the fatty acid moiety into the lipid bilayer; rather, they associate with specific receptors at the inner leaf of the plasma membrane. The specific association of MARCKS with focal contacts also suggests a receptor at the cytoplasmic face of the substrate-adherent plasma membrane. Mutational analysis suggests that the first 14 amino acids of MARCKS are essential for appropriate targeting, but since MacMARCKS and MARCKS differ in their first 20 amino acids, it its likely that they are targeted to different subcellular locations. However, given the similarity between the effector domains of MARCKS and MacMARCKS, it is likely that MacMARCKS also binds actin (Li et al, 1992).
MARCKS is widely distributed and has been implicated in cell motility, secretion, the regulation of the cell cycle, and transformation. MARCKS binds Ca calmodulin and F-actin, and this is regulated by phosphorylatlon. MARCKS cycles between the membrane and cytosol, and has been proposed to serve as a regulator of actin structure at the membrane, and of actin-membrane interactions. In contrast to MARCKS, MacMARCKS has a restricted distribution, and is mainly found in cells which have a high capacity for directed membrane traffic such as macrophages, neurons, and epithelial cells. Although MARCKs and MacMARCKs have a similar domain structure, and bind calmodulin and actin, they clearly have distinct functions. While MARCKS associates with the apical surface of polarized epithelial cells, MacMARCKS is targeted to the basolateral surface. MARCKS cycles reversibly between the membrane and the cytosol, while MacMARCKS always remains associated with the membrane. MARCKS has a role in actin remodeling in motility and phagocytosis, while MacMARCKS is apparently associated with vesicular traffic, and the recruitment of membranes to phagosomes. Interestingly, unlike MARCKS, MacMARCKS is not expressed in neutrophils, consistent with the thesis that these proteins serve different functions during phagocytosis.
Phagocytosis is an ancient adaptation which allows lower organisms to ingest nutrients, and higher organisms to capture and sterilize pathogens, to remove senescent material, and remodel tissues. Macrophages, monocytes, and neutrophils are considered "professional" phagocytes because of their dedication to this task. After internalization, phagosomes mature, ultimately fusing with lysosomes. Actin is required for phagocytosis, and perhaps has a role in regulating phagosome-lysosome fusion by temporally controlling access of lysosomes to the phagosomal membrane. However, the signal transduction pathways which regulate phagocytosis, and particularly phagosome-lysosome fusion, are obscure. Ca.sup.2+ has been implicated as a regulator of phagosome-lysosome fusion, and activated PKC has been shown to associate with the phagosome. Since MacMARCKS associates with phagosomes just before phagosome-lysosome fusion, and since its activity is regulated by Ca.sup.2+ /calmodulin and PKC, it is a good candidate molecule to integrate Ca.sup.2+ and PKC dependent signals in controlling phagosome-lysosome fusion.
Many microorganisms evade killing by circumventing specific steps in the phagocytic pathway. For example, Salmonella typhimurium enters macrophages via a spacious phagosoine which resembles a macropinosome. Although this structure subsequently fuses with lysosomes, its acidification is attenuated. In contrast, the IgG opsonized bacterium, and certain PhoP mutants of S. typhimuriuin, enter macrophages in phagosomes in which the membrane is tightly apposed to the particle. MacMARCKS associates with tight phagosomes containing dead S. typhimurium, or avirulent S. minnesota, but not with spacious phagosomes containing the virulent S. typhimurium. The relationship between MacMARCKS, virulence, and phagosome morphology will be investigated.
Transcytosis in polarized epithelial cells: Polarized epithelial cells play key roles in immune defense in addition to their barrier function; they both deliver antigens to the mucosal immune system, and export immunoglobulins into secretions that bathe the epithelial surfaces of mucosal tissues. Thus transcytosis across polarized epithelial cells constitutes a major limb of host response to infection. The best characterized examples of transcytosis involve the transport of immunoglobulins (Ig) across epithelia by the polymeric Ig receptor. The newly synthesized receptor is sorted to the basolateral surface, where it binds Pig, is endocytosed, transcytosed, and exocytosed at the apical surface. Little is known about the signaling systems which regulate transcytosis of the pIgR, although PKC and Ca.sup.2+ /calmodulin have been implicated. MacMARCKS is a PKC substrate which binds Ca.sup.2+ /calmodulin and which translocates from the basolateral to the apical surface of epithelial cells when phosphorylated. It therefore represents an excellent candidate as a regulator of transcytosis in epithelial cells.
MARCKS and MacMARCKS bear strong functional similarity to a neurospecific PKC substrate known as GAP-43 or neuromodulin (Liu et al (1990) Trends Pharmacol. Sci. 11:107-111). In contrast to MARCKS and MacMARCKS, which bind calmodulin in a calcium-dependent manner, GAP-43 associates with calmodulin in the absence of calcium. Moreover, unlike MARCKS and MacMARCKS, which are myristoylated at their N-termini, GAP-43 is palmitoylated. Both MARCKS and GAP-43 appear to have a role in regulating the motile cytoskeleton. GAP-43 associates with the actin cytoskeleton in neuronal growth cones and is a member of a family of proteins which includes neurogranin, a smaller protein which also contains a phosphorylation domain and calmodulin-binding site.
Another related PKC substrate is adducin, a protein which promotes the association of actin with spectrin in a calmodulin-regulated manner. Adducin has both structural and functional similarities to MARCKS and MacMARCKS (Joshi et al (1991) J. Cell Biol. 115:665-675). The protein is composed of highly homologous .alpha. and .beta. subunits; both subunits contain identical stretches of 22 amino acids in their C-termini with sequence similarity to the effector domains of MARCKS and MacMARCKS (Joshi et al, 1991). The C-termini of the .alpha. and .beta. subunits of adducin also bear the PKC phosplhorylation sites and the domain that binds calcium-calmodulin (Joshi et al, 1991).
Moreover, the following observations indicate that MacMARCKS and members of the MARCKS family of PKC substrates have a role in regulating cell movement and membrane traffic: (1) the effector domain regulates actin crosslinking and calcium-calmodulin binding; (2) the myristoylation domain mediates membrane binding; and (3) the MH2 domain has a role in subcellular targeting.
Little is known about the signal transduction pathways involved in mediating neural tube formation and closure, but studies with drugs implicate both the microtubules and microfilaments. It would thus be advantageous to analyze the role of MacMARCKS in such regulatory processes.